AU2020103364A4 - Ositinib-resistant cell line NCI-H1975/AR and application thereof - Google Patents
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Abstract
The invention discloses an ositinib resistant cell line NCI-H1975/AR and an
application thereof. The drug-resistant cell line is a non-small cell lung cancer drug
resistant cell line to AZD9291, namely, a human lung adenocarcinoma cell NCI
H1975/AR, which has been deposited in the China Typical Culture Collection Center
on June 14, 2019 with the deposit number CCTCC NO: C201982. The drug resistance
multiple of the drug-resistant strain is more than 10 times, It can stably grow and pass
in the culture system with the action concentration of 100nmol AZD9291, and the
drug resistance multiple (RI) to AZD9291 is 17.7 times. It provides a drug-resistant
cell model for studying the mechanism of drug resistance to targeted drugs in non
small cell lung cancer, finding effective therapeutic methods to overcome drug
resistance in non-small cell lung cancer and guiding clinical medication, and has
important role and good application prospect.
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H1975-PT H1975-AR
Figure 1
Description
-1/3
H1975-PT H1975-AR
Figure 1
Ositinib-resistant cell line NCI-H1975/AR and application thereof
The invention belongs to the technical field of biomedicine. More particularly, it relates to an ositinib-resistant cell line NCI-H1975/AR and applications thereof.
Lung cancer is the leading cancer mortality rate in the world. Non-small-cell lung cancer (NSCLC) accounts for 80% of lung cancer. The 5-year survival rate of non-small cell lung cancer is less than 15%, mainly because about 70% of patients are in advanced stage when they are first diagnosed with non-small cell lung cancer, and most of them have distant metastasis. Lung cancer is one of the first types of cancer to apply targeted therapy in clinical practice. In the past ten years, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has been an important drug for the treatment of advanced non-small cell lung cancer (NSCLC), and its target is epidermal growth factor receptor (EGFR). This kind of drug has the characteristics of definite curative effect, slight adverse reactions and convenient oral administration, which breaks through the bottleneck of traditional chemotherapy drugs.
At present, the first generation of EGFR-TKI used clinically include erlotinib (trade name Trocquer), gefitinib (trade name Iressa) and exitinib (trade name Kamina). The first generation of TKI is a reversible EGFR-RTK inhibitor, mainly targeting two common mutations (exon 19 deletion mutation and exon 21 L858R mutation). The second generation of EGFR-RTK has alfatinib, which is indicative of the same generation of EGFR-TKI, but is an irreversible EGFR inhibitor, mainly targeting rare mutation sites such as G719X, L861Q and S7681 of EGFR. The third generation and subsequent EGFR-TKI include compounds such as AZD9291 and CO-1686, which irreversibly inhibit EGFR, also have inhibitory effect on wild-type EGFR, and still have high effective rate for patients with T790M drug-resistant mutation. Although the first and second generations of targeted drugs have remarkable curative effects, 2/3 of the patients will develop drug resistance after 1-2 years of drug use, and the tumor may begin to rebound. The causes of drug resistance to targeted drugs vary 50 from patient to patient, but % ~ 60% of EGFR inhibitor resistance is related to T790M mutation. AZD9291 (ositinib) is an oral small molecule third generation EGFR-TKI. It is the first lung cancer drug targeting EGFR T790M mutation. It can target EGFR gene mutation (including 18, 19, 21 mutations) and EGFR-TKI acquired drug resistance (T790M) in non small cell lung cancer. AZD9291 (ositinib) has achieved remarkable curative effect in non small cell lung cancer.
However, after long-term use of AZD9291 (ositinib) targeted therapy, almost all patients will develop drug resistance. Therefore, it is of great significance to simulate the drug resistance process of NSCLC lung cancer AZD9291 and to deeply understand the drug resistance mechanism of NSCLC AZD9291. At present, many drug-resistant cell lines for tumors have been established at home and abroad, but there is no first-line drug-resistant cell line for non-small cell lung cancer to AZD9291.
Contents of the invention
The invention aims to provide a non-small cell lung cancer cell line resistant to the third generation EGFR-TKI drug ositinib (AZD9291).
The object of the invention is to provide a human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR resistant to the third generation EGFR-TKI drug ositinib (AZD9291).
A further object of that present invention is to provide an application of the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR in screen a drug for reversing tumor drug resistance.
A further object of that present invention is to provide the use of the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR in the preparation of an anti-tumor drug.
The above object of that invention is realize by the following technical scheme:
According to the invention, non-small cell lung cancer cells are taken as induction objects, and 100nmol of drugs are adopted to continuously treat non-small cell lung cancer cells in logarithmic growth period, and induction, domestication and culture are carried out for 6 months; Through cell morphological observation, drug sensitivity and drug resistance tests, To evaluate the biological characteristics of non-small cell lung cancer in AZD9291 cell line, A non-small cell lung cancer H1975/AZD9291 drug-resistant strain carrying drug resistant mutation T790M and EGFR 19 exon deletion was successfully constructed, named as human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR, i.e. Human lung adenocarcinoma cell NCI-H1975/AR, and deposited in China Typical Culture Collection Center on June 14, 2019 with the preservation number CCTCC NO: C201982 and the preservation address: Wuhan University, Wuhan City, China.
The drug-resistant multiple of the non-small cell lung cancer constructed by the invention to the AZD9291 drug-resistant cell strain is more than 10 times, It can stably grow and pass in the culture system with the action concentration of 100nmol AZD9291, and the drug resistance multiple (RI) to AZD9291 is 67 times. It provides a drug-resistant cell model for studying the mechanism of drug resistance to targeted drugs in non-small cell lung cancer, finding effective therapeutic methods to overcome drug resistance in non-small cell lung cancer and guiding clinical medication, and has important role and good application prospect.
Therefore, the application of the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR in screening drugs for reversing tumor drug resistance and in preparing anti tumor drugs should be within the scope of protection of the present invention.
Preferably wherein the tumor is lung cancer. Specifically, it refers to non-small cell carcinoma. More specifically lung adenocarcinoma.
The invention has the following beneficial effects:
A non-small cell lung canc drug resistant cell strain to AZD9291 is constructed, The drug resistance ratio is more than 10 times, It can stably grow and pass in the culture system with the action concentration of 100nmol AZD9291, and the drug resistance multiple (RI) to AZD9291 is 67 times. It provides a drug-resistant cell model for studying the mechanism of drug resistance to targeted drugs in non-small cell lung cancer, finding effective therapeutic methods to overcome drug resistance in non-small cell lung cancer and guiding clinical medication, and has important role and good application prospect.
Description of the drawings
FIGURE. 1 show that morphological changes of drug-resistant cell line; H1975/AR cells: The whole cell is slightly larger, and most of them are polygonal pseudopods.
FIGURE. 2 is the detection results of drug sensitivity and drug resistance of drug resistant cell lines. IC50: H1975/pt (parent strain): 0.05 0.01 M; H1975/AR (resistant strain): 3.35 0. 1MRI: 67 (highly resistant strain).
FIGURE. 3 is a comparison before and after gene sequencing of drug-resistant cell lines. H1975/pt was before drug resistance and H1975/AR was after drug resistance.
BRIEF DESCRIPTION OF THE DRAWINGS that present invention will be further describe below in connection with the accompanying drawing and specific embodiments of the specification, but the embodiments do not in any way limit the invention. Unless otherwise specified, the reagents, methods and apparatus employed in the present invention are conventional reagents, methods and apparatus in the art.
Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
EXAMPLE 1 induction and establishment of drug-resistant cell line
Induction and Construction of Human Lung Adenocarcinoma Drug Resistant Cell Line NCI-H1975/AR:
(1) NSCLC cell H1975 was cultured in a medium containing 10% inactivated fetal bovine serum and 1% double antibodies (1OOU/mlpenicillin and1OOg/ml streptomycin), and cultured in an electrothermal constant temperature incubator at 37 °C, 5% C02 and 96% humidity.
(2) NSCLC cells in logarithmic growth phase were inoculated in culture flask at 5 x 10 /mL, When cultured to 75% adherence rate, The cells were cultured in culture medium containing 100nM AZD9291 and cultured at 37 °C, 5% CO 2 and 96% humidity. The cells were cultured in culture medium containing AZD9291. The cells were cultured in culture medium containing AZD9291. The cells were cultured in culture medium containing AZD9291 at the concentration of 1 x 10 5/ml and washed once with 2ml PBS. The culture medium was cultured in culture medium containing AZD9291 at 37 °C, 5% CO 2 and 96% humidity. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. The culture medium was cultured in culture medium containing AZD9291. Replace the culture medium once every 2-3 days, after the cells resume growth and gradually grow into a petri dish, subculture the cells according to a ratio of 1: 3, and continuously use AZD9291 (100nM) for culture; The IC50 value was detected by MTT method every two weeks until a stable IC50 value was measured, and the drug-resistant strain was obtained when the IC50 value was more than 10 times that of the primary cell. After 6 months of culture, the cell lines growing stably and passaging in the culture system with 100nmol AZD9291 as the action concentration were screened, and the results showed that the cell lines were stable and passaged in the culture system with100nmol AZD9291 as the action concentration.
The obtained cell line was named as human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR, and was deposited in China Typical Culture Collection Center on June 14, 2019 with the deposit number CCTCC NO: C201982 and the deposit address: Wuhan University, Wuhan City, China.
EXAMPLE 2 Morphological observation of drug-resistant cell strain
1. Test cell line materials:
The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR, and the parent non small cell lung cancer cell line H1975/pt.
2. Test method:
The two kinds of cells were inoculated into 6-well plates respectively, cultured to logarithmic growth period, and the cell morphology was observed under inverted phase contrast microscope.
3. The test results are shown in Figure 1.
H1975/pt and H1975/AR cells grew in epithelium-like monolayer arrangement and adherence under microscopy. H1975/pt cells had clear boundaries, mostly long spindle shaped and even polygonal, with loose growth and voids. However, the volume of H1975/AR cells after drug resistance became larger, the boundary was unclear, and they gathered and grew in clusters. Most of the cells were polygonal and pseudopodia were formed.
Compared with parent cells, lung cancer drug-resistant cell lines are enlarged in volume, easy to form clusters and irregular in shape. This indicates that the drug-resistant cell line of tube cancer has changed in morphology.
EXAMPLE 3 detection of drug sensitivity and drug resistance of drug-resistant cell strain
1. Test cell line materials:
The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR, and the parent non small cell lung cancer cell line H1975/pt.
2. Test method:
The logarithmic non-small cell lung cancer resistant cell line H1975/AR and the parent non-small cell lung cancer cell line H1975/pt were prepared into cell suspension with a concentration of 30000 cells/ml respectively, and inoculated into 96-well cell culture plate with 2001 cell suspension (6000 cells) added to each well. The 96-well cell culture plate was placed in an incubator with 37 °C, 5% C02 and 96% humidity. After the cells were cultured for 24 hours and adhered to the wall, the old culture medium was carefully sucked out. 2001 of different concentrations of drug-containing culture medium solution was added to each well (the concentration gradient of H1975 cell AZD9291 was 0.01, 0.1, 0.5, 1, 3, 5, 6, 7, 8uM/ml). Six multiple pores were set up at each concentration. The negative control group was cultured with DMSO-containing culture medium. After 48 hours of continuous culture in the incubator, the old culture medium was carefully sucked out. 200ul of MTT solution (MTT: PBS = 1: 9) was added to each well, cultured at 37 °C for 4 hours, 150L of dimethyl sulfoxide (DMSO) was added, and mixed evenly in a shaking table for 10 minutes. The OD value of each well was measured by enzyme labeling instrument at = 490nm, and the inhibition rate and drug resistance index were calculated.
Inhibition rate (%) = (OD value of control group-OD value of test group)/OD value of control group x 100%
Drug resistance index (RI) = IC50 of drug-resistant cells/IC50 of parent cells.
3. Test Results:
The test results are shown in FIG. 2. The drug resistance index (RI) of the non-small cell lung cancer cell H1975/AR cell strain constructed by the invention to AZD9291 is 67 times.
EXAMPLE 4 detection of gene mutation in drug-resistant cell strain
1. Test cell line materials:
The non-small cell lung cancer drug-resistant cell line constructed in Example 1, namely the human lung adenocarcinoma drug-resistant cell line NCI-H1975/AR, and the parent non small cell lung cancer cell line H1975/pt.
2. Test method:
Illumina cell genome high-throughput sequencing and new generation sequencing (NGS) technology analyze the whole genome, which can provide base views of all genome changes, including single nucleotide site variation (SNV), insertion and deletion, copy number change and structural variation.
3. The test results are shown in Fig. 3.
H1975/pt cells: 16% interchromosomal translocation (CTX), 2% intra-chromosomal translocation (ITX) and 0% inversion (INV); Delete (DEL) 60%; Insertion (INS) 22%.
H1975/AR cells: 5% interchromosomal translocation (CTX), 12% intra-chromosomal translocation (ITX) and 0% inversion (INV); Delete (DEL) 71%; Insertion (INS) 12%.
Sequencing results show that the genetic background of H1975/AR cell has completely changed compared with H1975/pt cell, and it is a new cell line.
The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present invention shall be equivalent substitutions and shall be included in the scope of protection of the present invention.
Claims (6)
1. An ositinib-resistant cell line, characterize in that that ositinib-resistant cell line is a human lung adenocarcinoma cell NCI-H1975/AR, which was deposited in the China typical culture collection center on June 14, 2019 under the accession number CCTCC NO: C201982.
2. That use of an ositinib-resistant cell line accord to claim 1 for screening a drug for reverse tumor resistance.
3. That use of an ositinib-resistant cell line accord to claim 1 in the preparation of an anti-tumor drug.
4. That application accord to claim 2 or 3, wherein the tumor is lung cancer.
5. That use of claim 4, wherein the lung canc is a non-small cell carcinoma.
6. That method accord to claim 5, wherein the lung cancer is a lung adenocarcinoma.
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Figure 2
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Figure 3
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Oxitinib drug-resistant cell strain NCI-H1975/AR and application thereof |
| CN115927187A (en) * | 2022-12-30 | 2023-04-07 | 河南省肿瘤医院 | Ametinib drug-resistant cell line NCI-H1975-AR and application thereof |
| CN116200340A (en) * | 2022-12-30 | 2023-06-02 | 河南省肿瘤医院 | Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof |
| CN116355851A (en) * | 2023-03-13 | 2023-06-30 | 广州医科大学附属第一医院（广州呼吸中心） | Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof |
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2020
- 2020-11-10 AU AU2020103364A patent/AU2020103364A4/en active Active
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Oxitinib drug-resistant cell strain NCI-H1975/AR and application thereof |
| CN111218424B (en) * | 2019-12-12 | 2022-04-15 | 广州医科大学附属第一医院 | Oxitinib drug-resistant cell strain NCI-H1975/AR and application thereof |
| CN115927187A (en) * | 2022-12-30 | 2023-04-07 | 河南省肿瘤医院 | Ametinib drug-resistant cell line NCI-H1975-AR and application thereof |
| CN116200340A (en) * | 2022-12-30 | 2023-06-02 | 河南省肿瘤医院 | Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof |
| CN116200340B (en) * | 2022-12-30 | 2023-08-15 | 河南省肿瘤医院 | Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof |
| CN115927187B (en) * | 2022-12-30 | 2024-01-23 | 河南省肿瘤医院 | Ameitinib resistant cell strain NCI-H1975-AR and application thereof |
| CN116355851A (en) * | 2023-03-13 | 2023-06-30 | 广州医科大学附属第一医院（广州呼吸中心） | Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof |
| CN116355851B (en) * | 2023-03-13 | 2023-09-08 | 广州医科大学附属第一医院（广州呼吸中心） | Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof |
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